Abstract Studies from our laboratory over the past 16 years have provided insights into the molecular basis for human trophoblast differentiation and the mechanisms by which committed progenitor cells differentiate into endocrine effector cells. We have shown that the transcription factor AP-2a is essential for biochemical differentiation as based on the induction of placental lactogen, chorionic gonadotropin and other syncytiotrophoblast-specific genes. Morphological differentiation is dependent on the action of syncytin, a transmembrane glycoprotein that is induced during villous cytotrophoblast differentiation in an AP-2a- independent manner. Our overall hypothesis is that the activation of AP-2a is essential for trophoblast differentiation and that transcription factors and signaling molecules that induce AP-2a expression and function, along with downstream targets of AP-2a action, comprise a regulatory network critical for the differentiation of cytotrophoblast cells to syncytiotrophoblast cells. Specific Aim 1 will test the hypothesis that transcription factors and signal transduction molecules that induce syncytin gene expression and cell fusion are early components of the regulatory pathway that leads to the induction of AP-2a and the terminal differentiation of CTB to a STB phenotype. Specific Aim 2 will test the hypothesis that AP-2a modulates biochemical differentiation by regulating the expression of key downstream genes involved in the accomplishment of cell and tissue structure, extracellular matrix formation, inhibition of cellular proliferation and apoptosis. Specific Aim 3 will test the hypothesis that the AP-2a protein is phosphorylated during CTB differentiation and forms novel complexes with other transcription factors and co-activators that recognize stereotypical cis-regulatory element modules within STB and CTB enhancers and promoters. Methods include transduction of isolated human cytotrophoblast cells with lentiviruses that express specific shRNAs or genes, genome-wide DNA microarrays, ChIP-chip experiments, and co-immunoprecipitation studies. Taken together, the results will provide new integrated insights into the mechanism of placenta development and function, novel understanding of the regulation of the determination and differentiation of a highly competent human somatic progenitor cell, and a set of broadly useful data that will enable subsequent detailed studies by investigators in diverse research areas. Comprehensive knowledge of the regulatory network that directs cytotrophoblast differentiation should provide new insights into the pathogenesis of pathologic conditions of pregnancy associated with defective placentation, such as pre-eclampsia and choriocarcinoma.